This invention relates to reagents for diluting and lysing whole blood particularly employed in automatic instruments for blood particle counting and hemoglobin determination.
In relatively simple blood particle counting instruments, for example, the HEMO-W.RTM. instrument manufactured by Coulter Electronics, Inc. of Hialeah, Fla., measurements are limited to the simultaneous determination of hemoglobin concentration and total leukocyte count of a whole-blood specimen. In such a system, the erythrocytes are lysed (hemolyzed) to liberate their hemoglobin for quantitative determination. The white blood cells (WBC) remain as individually countable particles suited to the Coulter.RTM. electronic-impedance method.
An individual blood sample is prepared, manually or in an automatic diluter, by addition to a combined diluting and lysing solution before aspiration into the HEMO-W.RTM. instrument. Prior combined diluting and lysing reagents, for example, Isolyse.RTM. manufactured by the Coulter Diagnostics Division of Coulter Electronics, Inc. have included the conventional disodium salt of ethylenadiaminetetraacetic acid (EDTA) to inhibit platelet aggregation as well as to prevent coagulation of the sample. In some instances, erroneously high WBC count has been observed with the HEMO-W.RTM. instrument because platelet aggregations have been included in the WBC count. This problem has occurred with capillary blood samples which are obtained in small quantity by the "finger stick" method and then directly added to the combined diluting and lysing reagent in which lysing begins immediately. Artificially elevated WBC count has not occurred using venous blood samples which are conventionally added directly to a diluent and anticoagulant solution containing EDTA, prior to further addition to the combined diluting and lysing solution for aspiration into the instrument. It has been determined that the EDTA component in existing formulations of combined diluting and lysing solutions is incapable of preventing platelet aggregation under the conditions of concurrent lysing, unless the blood sample has been previously subjected to an anticoagulation agent. Once lysing of the sample has occurred, platelet aggregates will not disperse and addition of further EDTA merely promotes cohesion of the aggregates.
Multi-purpose blood diluents for use in automatic hematology instruments have been described in U.S. Pat. No. 3,962,125, U.S. Pat. No. 4,213,876 and U.S. Pat. No. 4,346,018. The multi-purpose diluents described in these patents do not include lysing agents, and are specifically formulated to stablize and preserve the red blood cells so that their volume can be accurately measured and counted in addition to counting of white blood cells. U.S. Pat. No. 4,213,876 describes the use of inorganic metalic sulfate for suppressing turbidity caused by abnormal plasma globulins or by elevated white blood cell count which can affect hemoglobin determinations.
U.S. Pat. Nos. 3,874,852 and 4,346,018 describe lysing reagents which are formulated for lysing samples which have been previously diluted for counting of red blood cells and white blood cells.